Around 1 to 15% of patients treated by radiotherapy for cancer have a tissue reaction (such as dermatitis or proctitis), which may hinder the treatment insofar as it may lead the physician to decide to stop the radiotherapy treatment before the end of the planned protocol. Moreover, this tissue reaction is an indicator of a particularly high sensitivity of the patient to ionizing radiation. Thus, the radiotherapy treatment, even if interrupted upon the appearance of the first visible tissue signs, may increase morbidity or even post-treatment mortality of patients, not only because the cancer that was supposed to be treated could not be fully eradicated due to the premature discontinuation of treatment but due to collateral damage to healthy tissue caused by the radiation itself.
It is also known that the question of sensitivity of tissue to ionizing radiation is inseparable from those of DNA damage repair mechanisms. In fact, at the cellular level, ionizing radiation may break certain types of chemical bonds, producing free radicals (in particular by peroxidation) and other reactive species produced by DNA damage. DNA damage by endogenous or exogenous stress (such as ionizing radiation and free radicals), may lead to different types of DNA damage in particular according to the energy applied: base damage, single-strand breaks and double-strand breaks (DSB). Non-repaired DSBs are associated with cell death, toxicity and more specifically radiosensitivity. Poorly repaired DSBs are associated with genomic instability, mutagenic phenomena and predisposition to cancer. The organism has systems for repair specific to each type of DNA damage. Concerning DSBs, mammals have two main modes of repair: repair by suture (strand ligation) and repair by recombination (insertion of a homologous or non-homologous strand).
It is known that sensitivity of tissues to ionizing radiation is highly variable from one organ to another and from one individual to another; the idea of “intrinsic radiosensitivity” was conceptualized by Fertil and Malaise in 1981. Thus, the various studies on the therapeutic effects and the adverse effects of radiotherapy have demonstrated that there are individuals with particularly high radioresistance, and individuals who, by contrast, have radiosensitivity that may range from a clinically recognized but inconsequential adverse effect to a lethal effect. Even outside of certain rare cases of extreme radiosensitivity, the genetic origin of which appears to be recognized, it is thought that radiosensitivity is generally based on a genetic predisposition: it is therefore specific to an individual. It would therefore be desirable to have a predictive test method in order to determine the maximum cumulative dose that a given patient may receive without risk. This question is posed first in radiotherapy in a context of high ionizing doses. However, this question may also be posed for any other exposure to high ionizing doses, equivalent to those used in radiotherapy.
It is known that two proteins of the kinase family, commonly called ATM and ATR, are involved in the detection, repair and signaling of DSBs; their action requires at least the presence of a protein known as BRCA1 and an ordered cascade of phosphorylations of the different ATM substrates (see article of N. Foray et al., “A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein”, published in The EMBO Journal vol. 22(11), p. 2860-2871 (2003)). It has been attempted to use the ATM enzyme in an explanatory model of cell radiosensitivity (see Joubert et al., “DNA double-strand break repair defects in syndromes associated with acute radiation response; At least two different assays to predict intrinsic radiosensitivity?”, published in Int. J. Radiat. Biol., vol. 84(2), p. 107-125 (2008)), and this made it possible to identify three types of radiosensitivity: radioresistant cells (Group I radiosensitivity), moderately radiosensitive cells (Group II radiosensitivity, and extremely radiosensitive cells (Group III radiosensitivity). However, no predictive model was proposed on this basis, in particular no relationship was established between the clinical data (tissue severity grade) and the radiobiological data. Similarly, the presentation of N. Foray, “Les réparatoses: nouveaux concepts sur la prédiction de la radiosensibilité”, presented at “Rencontres Nucléaire & Santé” on Jan. 25, 2008 (XP55131242), suggests the role of the different markers pH2AX and MRE11 and the changes thereof over time for describing the number of radiation-induced double-strand breaks. This presentation does not mention the tissue severity grades, which quantify and identify the degree of radiosensitivity observed at the clinical level.
Numerous documents describe the conditions in which ATM may contribute to the detection and repair of DNA damage. The patent application WO 2004/013634 (KUDOS Pharmaceuticals Ltd.) describes the identification of an ATM-dependent DNA damage-signaling pathway that interacts with other DNA damage response factors, including the MRE11/Rad51/NBS1 complex. The patent application US 2007/0072210 (Ouchi and Aglipay) proposes a method for screening potential therapeutic agents that promotes a response to DNA damage, in which a protein called BAAT1 (that is associated with a predisposition to cancer associated with the BRCA1 gene), an ATM protein and the candidate compound are mixed; if the phosphorylation of ATM is increased with respect to a control mixture not containing the candidate compound, the latter is identified as being a potential active principle promoting DNA repair. The patent application EP 2 466 310 A1 (Helmholtz Zentrum München) describes the repair of DNA double-strand breaks in the presence of the phosphorylated form of H2AX histone (called gamma-H2AX or g-H2AX). The application WO 00/47760 and U.S. Pat. No. 7,279,290 (St. Jude's Children's Research Hospital) describe the role of the ATM kinase function in DNA repair.
These last documents therefore describe repair pathways but do not offer any correlation for establishing a clinical link.
Patent EP 1 616 011 B1 (International Centre for Genetic Engineering and Biotechnology) proposes a method for diagnosis of a genetic defect in DNA repair based on three steps: the culture of isolated cells of a sample to be tested, the incubation of said cells with a chimeric polypeptide, the characterization of the cell response. Said cell response is the expression level of a biochemical marker consisting of the following types of intracellular proteins: p53, ATM, Chk1, Chk2, BRCA1, BRCA2, Nbs1, MRE11, Rad50, Rad51 and histones. However, the radiation-induced expression cannot be predictive of the functionality of said proteins (certain syndromes have a normal expression level when the protein is mutated): these procedures are not functional tests.
The patent applications WO 01/90408, WO 2004/059004 and WO 2006/136686 (French Atomic Energy Commission) describe other methods for observing DNA damage resulting from ionizing radiation. The first document concerns the demonstration of DNA lesion incision activities, and does not enable the quantification of enzymatic activities of excision and re-synthesis of DNA or DSB repair. The two other documents describe the quantitative evaluation of the capacities of a biological medium to repair its DNA by means of circular supercoiled double-strand DNA (according to the third document: immobilized in a porous polyacrylamide hydrogel film). These methods do not directly concern DSBs in their in situ physiological environment and there is no correlation for validating their clinical application.
KR20030033519 proposes deducing sensitivity to radiation from the catalysis or superoxide dismutase activity, and KR20030033518 uses glutathione peroxidase or glucose 6-phosphate dehydrogenase. Such methods do not detect markers directly linked to DNA damage or repair.
Patent application US 2011/312514 (Dana Farber Cancer Institute) proposes using the detection of FANCD2 foci as a marker. Patent application US 2007/0264648 (National Institute of Radiological Sciences) proposes the use of DNA oligomers for predicting the appearance of adverse effects in radiotherapy. However, certain kinds of radiosensitivity may be observed when the FANCD2 foci level is normal.
Patent applications US 2008/234946 and US 2012/041908 (University of South Florida et al.) describe a method for predicting radiosensitivity of cancer cells and not healthy cells; it is also based on genomic data and not functional tests.
Patent application WO 2014/154854 (Centre Hospitalier Universitaire de Montpellier) describes a method for predicting radiosensitivity of a subject via the use of at least one radiosensitivity biomarker. This method does not detect markers directly linked to DNA damage or repair; it is also based on proteomic data. Moreover, this patent application does not describe a quantitative relationship between the radiobiological data and the severity of the tissue reactions.
Patent application WO 2013/187973 (University of California) describes systems and methods for determining the radiosensitivity of cells and/or of a subject with regard to a control population. More specifically, this method includes the irradiation of a biological sample, the detection and quantification of radiation-induced foci in erythrocytes, lymphocytes and primer cells, resulting from a blood sample via the use of one or more detection markers among a set of markers including anti-pH2AX, anti-MRE11 and anti-ATM. The quantification of radiation-induced foci at different post-irradiation observation times below 2 hours makes it possible to determine their repair kinetics, empirically correlated with the radiosensitivity of the subject. However, the analysis of foci in lymphocytes is very difficult due to their small nucleus. Moreover, this method does not enable the practitioner to make decisions regarding the treatment of the patient.
U.S. Pat. No. 8,269,163 (New York University School of Medicine) describes a large number of proteins capable of being used as markers for simply and rapidly assessing the significance of accidental exposure of a person to ionizing radiation, in order to triage patients and direct them to the appropriate emergency treatment. This last patent concerns biological dosimetry (determination of the accidental dose) while the detection of radiosensitivity is performed on the basis of a known dose.
Patent application WO 2010/88650 (University of Texas) describes methods and compositions for identifying cancer cells that are sensitive or resistant to a particular radiotherapy treatment; therefore, they are not applicable to just any radiotherapy treatment.
Patent application WO 2010/136942 (Philips) describes a general method for monitoring a patient during radiotherapy by means of biomarkers. The method includes obtaining at least one descriptor based on an image extracted from an imaging modality, in which the descriptor belongs to a tissue of interest for which radiotherapy is provided or to a tissue in the vicinity of the target volume. The method also includes the selection of at least one biological markers specific to a disease, capable of detecting or quantifying adverse effects of radiotherapy in the area of the tissues of interest. In addition, the method includes the recovery of at least one in vitro measurement value of the selected biomarker specific to the disease. In addition, the method includes the treatment of the at least one descriptor of the at least one in vitro biomarker value by means of a correlation technique, resulting in an output signal indicating radiotoxicity in the region of the tissue of interest. However, the teaching of this patent takes into account only the tissue-dependent toxicity and not the individual, and is primarily based on image analysis.
Patent application WO 2010/109357 describes a method and a device for planning an adaptive radiotherapy protocol based on the optimization of the probability of normal tissue complication and the probability of tumor control according to markers specific to each patient. The values of the markers associated with normal tissues include in vitro test values, signatures by mass spectrometry of proteins, and data of the patient's history. The in vitro test values may be of cellular, proteomic and genetic origin, such as, but without being limited to, various cell counts, HB, CRP, PSA, TNF-alpha, ferritin, transferrin, LDH, IL-6, hepcidin, creatinine, glucose, HbAlc, and telomere length. The patient's history markers include prior abdominal surgery, hormonal drugs or anticoagulants, diabetes, age and measurements associated with tumor growth. Biomarkers not associated with radiotoxicity are also envisaged, such as biomarkers associated with various forms of ablation or chemotherapy agents. However, individual radiosensitivity is not taken into account.
In spite of this vast prior art, the applicant notes that the above-described patents do not describe a method for quantification of individual radiosensitivity making it possible to evaluate the risk of post-radiotherapy tissue reactions, which may be used for any patient and any type of ionizing radiation capable of inducing DSB, and which is predictive. The problem of providing a method predicting individual radiosensitivity therefore has not operational solution. This invention is intended to propose a new method for predicting tissue and clinical radiosensitivity.